Label-Free High-Throughput Leukemia Detection by Holographic Microscopy

Abstract

Complete blood count and differentiation of leukocytes (DIFF) belong to the most frequently performed laboratory diagnostic tests. Here, a flow cytometry-based method for label-free DIFF of untouched leukocytes by digital holographic microscopy on the rich phase contrast of peripheral leukocyte images, using highly controlled 2D hydrodynamic focusing conditions is reported. Principal component analysis of morphological characteristics of the reconstructed images allows classification of nine leukocyte types, in addition to different types of leukemia and demonstrates disappearance of acute myeloid leukemia cells in remission. To exclude confounding effects, the classification strategy is tested by the analysis of 20 blinded clinical samples. Here, 70% of the specimens are correctly classified with further 20% classifications close to a correct diagnosis. Taken together, the findings indicate a broad clinical applicability of the cytometry method for automated and reagent-free diagnosis of hematological disorders.

Keywords: digital holographic microscopy; label‐free detection; leukemia; microfluidics.

Figure 1

Microfluidics cell presentation and five‐part DIFF of leukocytes. a) Four sheath inlets (indicated as 1–4) form a 2D sheath flow around the sample (inlet 5). b) Schematic side view of the microfluidics channel shown in (a). A focused monolayer of enriched leukocytes is formed. c) Enlarged schematics of the sample flow shown in (b). The physical DOF of ±2.3 µm results in imaging of cell segments which pass the optical field in random positions. d) Purified leukocyte populations of multiple healthy donors separated by PCA. e) The combination of PCA component and entropy significantly improved the separation efficiency of eosinophils and neutrophils.

Figure 2

Label‐free, ungated single cell analysis allows leukemia subtyping. a) Label‐free, ungated density plots of healthy control and b) samples with acute myeloid leukemia (AML), c) acute lymphocytic leukemia (ALL), d) myeloproliferative neoplasm (MPN), e) special MPN chronic myelogenous leukemia (CML), and f) chronic lymphocytic leukemia (CLL). Parameters PCA4 and PCA5 are plotted. Each density plot shows representative single cell data from one sample. Percentages of cells are indicated for each plot quadrant.

Figure 3

Label‐free DIFF of eight leukocyte subtypes. Top row: reconstructed phase images of leukocyte subtypes. Bottom row: corresponding heat maps of top row phase images. Single cell data of multiple healthy and leukemic samples were used for the differentiation of leukocyte subtypes. Hematology analyzer data of healthy and blood smear analysis of leukemic samples were used as reference. Baso, basophil; Eos, eosinophil; Lympho, lymphocyte; Mono, monocyte; Neutro, neutrophil; MM/MY, meta‐/myelocyte; PM, promyelocyte. Scale bars are 5 µm.

Figure 4

Differentiation of ALL and CLL from AML and MPN. b) Acute lymphocytic leukemia (ALL) and c) chronic lymphocytic leukemia (CLL) are differentiated from a) acute myeloid leukemia (AML) and d) myeloproliferative neoplasm (MPN) by characteristic distribution of atypical lymphocytes (aL) and blasts (Bl(1)). Density plots show representative data of single samples. PCA components are plotted. Percentages of cells are indicated for each gate in the dot plots.

Figure 5

Gating strategy to differentiate AML and MPN. a) Gating strategy to differentiate acute myeloid leukemia (AML) from c) myeloproliferative neoplasm (MPN) and b) healthy control by characteristic distribution of neutrophils (Ne), immature granulocytes (IG), and blasts (Bl(2)). d) Chronic myelogenous leukemia (CML) is differentiated from other MPN by a unique scatter pattern. Gate IG & Bl = immature granulocytes and blasts. Density plots show PCA parameters and a combination of PCA and energy/entropy. Each dot plot shows representative data from a single sample. Percentages of cells are indicated for each gate.

Figure 6

Progression of AML from diagnosis to remission. a,b) Ungated density plots of acute myeloid leukemia (AML) directly after first diagnosis and same patient in remission. c,d) Percentage distribution of neutrophils (Ne), immature granulocytes (IG), and blasts (Bl(2)) from diagnosis to remission. Gate IG & Bl = immature granulocytes and blasts. PCA parameters and a combination of PCA and morphological parameters energy/entropy are plotted. Percentages of cells are indicated for each gate or density plot quadrant.